The cholesterol-dependent cytolysins (CDCs) are produced and contribute to disease caused by a wide variety of Gram-positive pathogens from the streptococci, clostridia, Bacillus, Listeria and arcanobacteria. In the present renewal we will continue to pursue the structural biology of the pore-forming mechanism and continue our investigation into the basis of cellular recognition by the CDCs. In the first aim we will identify the monomer-monomer contacts within domain 1 of the CDC structure that are involved in setting the oligomer geometry. Specific mutations within domain 1 dramatically alter the oligomer size and geometry of the CDC oligomeric pore complex. These observations indicate that there are important monomer-monomer contacts that occur in domain 1 that are necessary for proper oligomer formation. In the second aim we will investigate an aspect of pore formation that is pertinent to all [unreadable]-barrel toxins: Do the transmembrane [unreadable]-hairpins form a pre-insertion [unreadable]-barrel immediately before they insert into the membrane? The energetic cost of inserting naked [unreadable]-hairpins into the membrane bilayer core is high due to the unsatisfied hydrogen bind potential of the [unreadable]-strands. The energetic cost could be reduced considerably if the [unreadable]-hairpins of the oligomer were to form a pre-insertion [unreadable]-barrel in the prepore complex. In this aim we will determine if the [unreadable]-hairpins of the individual monomers form a [unreadable]-barrel structure in the prepore complex. In the third aim we will elucidate the components of the CDC structure that are involved in the conformational coupling of the domain 4 undecapeptide and the domain 3 transmembrane [unreadable]-hairpins. We now have evidence that suggests that a series of ordered conformational changes are triggered by membrane insertion of the undecapeptide that ultimately lead to the insertion of the transmembrane [unreadable]-hairpins. In this aim we will decipher the structural pathway that links the structural state of the undecapeptide to that of the transmembrane [unreadable]-hairpins and thereby controls prepore to pore conversion. In the last aim we will further investigate the basis of cellular recognition by the CDCs. For decades it has been generally accepted that cholesterol serves as the CDC receptor, but we now believe that cholesterol-mediated binding may be an artifact. We have shown one CDC, intermedilysin (ILY) uses human CD59 as its receptor and that membrane cholesterol is necessary for prepore to pore conversion. Until now it was not clear if ILY was a paradigm for cellular recognition by the CDCs or whether its ability to bind a non-sterol receptor was unique. We now have strong evidence that the CDC from Listeria monocytogenes, listeriolysin 0 (LLO) uses one or more specific cellular protein receptors present on the membranes of zebrafish embryonic tissue. We will identify these putative receptors in the final aim of this proposal. The aims of this proposal will be achieved by a combination of site-specific mutagenesis, fluorescence spectroscopy, and various biochemical approaches. This proposal continues our long-term goal to gain a deeper understanding of the pore-forming mechanism of the CDC and how these toxins specifically recognize cells. These studies will provide a better understanding of how these toxins function at the cell surface and will eventually allow us to design antagonists that block the activity of the CDC that could help in the treatment of a wide variety of serious bacterial infections.